title: Novel sterol glucosyltransferase in the animal tissue and cultured cells: evidence that glucosylceramide as glucose donor.
abstract: Cholesteryl glucoside (CG), a membrane glycolipid, regulates heat shock response. CG is rapidly induced by heat shock before the activation of heat shock transcription factor 1 (HSF1) and production of heat shock protein 70 (HSP70), and the addition of CG in turn induces HSF1 activation and HSP70 production in human fibroblasts; thus, a reasonable correlation is that CG functions as a crucial lipid mediator in stress responses in the animal. In this study, we focused on a CG-synthesizing enzyme, animal sterol glucosyltransferase, which has not yet been identified. In this study, we describe a novel type of animal sterol glucosyltransferase in hog stomach and human fibroblasts (TIG-3) detected by a sensitive assay with a fluorescence-labeled substrate. The cationic requirement, inhibitor resistance, and substrate specificity of animal sterol glucosyltransferase were studied. Interestingly, animal sterol glucosyltransferase did not use uridine diphosphate glucose (UDP-glucose) as an immediate glucose donor, as has been shown in plants and fungi. Among the glycolipids tested in vitro, glucosylceramide (GlcCer) was the most effective substrate for CG formation in animal tissues and cultured cells. Using chemically synthesized [U-((13))C]Glc-β-Cer as a glucose donor, we confirmed by mass spectrometry that [U-((13))C]CG was synthesized in hog stomach homogenate. These results suggest that animal sterol glucosyltransferase transfers glucose moiety from GlcCer to cholesterol. Additionally, using GM-95, a mutant B16 melanoma cell line that does not express ceramide glucosyltransferase, we showed that GlcCer is an essential substrate for animal sterol glucosyltransferase in the cell.
authors.affiliation: Graduate School of Humanities and Sciences, Department of Life Science, Ochanomizu University, 2-1-1 Ohtsuka, Bunkyo-ku, Tokyo 112-8610, Japan.
authors.forename: Hisako
authors.initials: H
authors.lastname: Akiyama
authors.forename: Narie
authors.initials: N
authors.lastname: Sasaki
authors.forename: Shuwa
authors.initials: S
authors.lastname: Hanazawa
authors.forename: Mari
authors.initials: M
authors.lastname: Gotoh
authors.forename: Susumu
authors.initials: S
authors.lastname: Kobayashi
authors.forename: Yoshio
authors.initials: Y
authors.lastname: Hirabayashi
authors.forename: Kimiko
authors.initials: K
authors.lastname: Murakami-Murofushi
mesh_terms: D000818:Animals; D001681:Biological Assay / Q000379:methods; D002478:Cells, Cultured; D002784:Cholesterol / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism; D005347:Fibroblasts / Q000166:cytology / Q000201:enzymology; D005947:Glucose / Q000378:metabolism*; D005963:Glucosylceramides / Q000737:chemistry / Q000378:metabolism*; D005964:Glucosyltransferases / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D013261:Sterols / Q000378:metabolism*; D013270:Stomach / Q000033:anatomy & histology / Q000201:enzymology; D013552:Swine; D014530:Uridine Diphosphate / Q000378:metabolism
chemical_list: D005963:Glucosylceramides; D013261:Sterols; C027578:cholesteryl glucoside; D014530:Uridine Diphosphate; D002784:Cholesterol; D005964:Glucosyltransferases; D005947:Glucose